Anti-Spectrin (alpha + beta) antibody [B12G3] (ab33426)

Overview

  • Product name
    Anti-Spectrin (alpha + beta) antibody [B12G3]
  • Description
    Mouse monoclonal [B12G3] to Spectrin (alpha + beta)
  • Host species
    Mouse
  • Tested applications
    Suitable for: ELISA, IHC-Frmore details
  • Species reactivity
    Reacts with: Human
  • Immunogen

    Native beta spectrin.

Properties

Applications

Our Abpromise guarantee covers the use of ab33426 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
ELISA Use at an assay dependent dilution.
IHC-Fr Use at an assay dependent concentration.

Target

  • Relevance
    Spectrin is a major constituent of the erythrocyte skeleton accounting for 25% of the total membrane protein and 75% of the cytoskeletal mass. Spectrin is an actin crosslinking and molecular scaffold protein that links the plasma membrane to the actin cytoskeleton by associating with the cytoplasmic surface of the membrane by attachment to ankyrin. The membrane skeleton influences several cellular properties such as cell shape, restriction of mobility of the integral membrane protein exposed at the cell surface and transmembrane movement of phospholipids and cholesterol and organization of organelles. Spectrin is a tetramer made up of alpha-beta dimers linked in a head-to-head arrangement. Mutations in spectrin result in a variety of hereditary red blood cell disorders, including elliptocytosis type 2, pyropoikilocytosis, and spherocytic hemolytic anemia.
  • Cellular localization
    Cytoplasmic; Plasma membrane; Cytoskeleton
  • Database links
  • Alternative names
    • Alpha I spectrin antibody
    • Beta I spectrin antibody
    • Beta spectrin antibody
    • EL2 antibody
    • EL3 antibody
    • Erythroid Alpha Spectrin antibody
    • HPP antibody
    • HS2 antibody
    • HSPTB1 antibody
    • Spec A antibody
    • Spec B antibody
    • Spectrin Alpha Chain antibody
    • Spectrin Beta Chain antibody
    • Spectrin beta chain erythrocyte antibody
    • Spectrin beta erythrocytic antibody
    • spectrin, alpha, erythrocytic 1 (elliptocytosis 2) antibody
    • SPH2 antibody
    • SPH3 antibody
    • SPNA1 antibody
    • SPNB1 antibody
    • SPTA antibody
    • SPTA1 antibody
    • SPTB1 antibody
    see all

References

ab33426 has not yet been referenced specifically in any publications.

Customer reviews and Q&As

1-5 of 5 Abreviews or Q&A

Question
Answer

Thank you very much for sending the data over.

I am very happy to hear that the antibodies worked well for you! I am also very happy to see, that all the antibodies seem to provide the expected doublet band. For the ab11184 it is effectively not that easy to see - can you confirm that there is also a doublet for ab11184?

As for the Western blot images for the feedback, naturally the better the images the more useful the feedback is. However if you write a comment maybe, describing what you found (i.e. the two bands at 1/500 for example), this will be already be very useful for us! So please do not hesitate to provide us the feedback like it is, and if one day you have the opportunity to make better images, we would obviously be pleased to receive them and replace these images.

I hope this is how you would like to proceed as well. Please let me know if you have any questions.

I wish you a great week!

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Answer

Thank you for your reply.

I am happy to hear that the antibodyprovides a signal after optimisation, even though it is not very strong. I do not know whether this activity is due to the vial, the batch or if it is intrinsec of the antibody itself. The ab33426 is an IgM antibody. IgM has in general lower affinity than IgG and I would therefore think and expect that this antibody needs to be used at a higher concentration then an IgG antibody.

As you remember correctly, we do not have another antibody against spectrin (alpha + beta) which has already been tested in ELISA. We do have two antibodies, the polyclonal ab11182 and the monoclonal ab11184 which have both been tested in Western blot and Immunocytochemistry.Wouldyoulike to test one of these in ELISA?
https://www.abcam.com/index.html?datasheet=11184 (or use the following: https://www.abcam.com/index.html?datasheet=11184).
https://www.abcam.com/index.html?datasheet=11182 (or use the following: https://www.abcam.com/index.html?datasheet=11182).

I would be able to provide you with one of these for free, if you would agree to provide us then with the feedback on whether they worked in ELISA or not.

Please let me know how you would like to proceed. I am looking forward to your reply.

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Answer

I hope you had a good weekend.

Did you already had the opportunity to retest this antibody?

Indeed, I am writing to inquire if the suggestions have helped to improve the results? I would be pleased to receive an update on how these experiments are progressing.

If the antibody is still not working as stated on the datasheet, and the item was purchased within 6 months of contacting us, we would be happy to replace or refund the antibody.

I wish you the best of luck with your research and I look forward to hearing from you. Please do not hesitate to contact us if you have any further questions.

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Question

Here are the answers for the questionnaire you sent me.
1) Abcam product code ab 33426

2) Abcam order reference number or product batch number Lot GR32825-1
3) Description of the problem Low efficiency. Interacts with haemoglobin
4) Type of ELISA: direct
5) Sample details: Erythrocyte membrane extract run on gel filtration and / or DEAE and / or MONO Q
6) Target antigen (if found in heterogeneous sample) Spectrin
7) Positive control none
8) Negative control antibody only ELISA, both primary and secondary
9) Blocking agent (eg BSA…): Marvel (skimmed milk powder) and glycine
Concentration 5% milk
Blocking time 1 hr
Blocking temperature rm temp
10) Capture or Primary antibody information
Used to coat wells? no
Species
Reacts against
Concentration or dilution
Diluent buffer
Incubation time
Incubation temperature
11) Detection antibody:
Species Mouse
Reacts against human spectrin
Concentration or dilution 1:100 – 1:1000
Diluent buffer blocking buffer
Incubation time 2hr
Incubation temperature: 37oC
Conjugation none
12) Enzyme labeled secondary antibody:
Species goat anti mouse
Reacts against mouse primary antibody
Concentration or dilution 1:1000 – 1:5000
Diluent buffer blocking buffer
Incubation time 2hr
Incubation temperature: 37oC
Conjugation HRP
13) Washing after primary and secondary antibodies:
Buffer PBST (0.02% v/v Tween-20)
Number of washes 3
Length of washes flood for a few seconds
14) Substrate
Incubation time 10-30 mins
15) How many times have you run this ELISA? 5
Do you obtain the same results every time? No
What steps have you altered to try and optimize the use of this antibody? Primary antibody conc, and secondary antibody conc. concentrate up the protein.

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Answer

Thank you for taking time to complete our questionnaire. I am sorry that this antibody is not providing satisfactory results.

The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality.

We do have different antibodies against spectrin, however this antibody is the only we have tested so far in ELISA. If you would like to test another one, please do let me know and I will be happy to see what testing offer we can make. I would however appreciate first, to try to solvethe problem you are experiencing with the ab33426.
Asyou know, inthe event that a product is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund.

Having reviewed this case, I would like to offer some suggestions to help optimize the results from ab33426. I would also appreciate if you can confirm some further details:

1.) The blocking buffer is crucial for a good result in ELISA. I would therefore recommend to use pure BSA for blocking. In my experience, pure BSA provided the best results. I can recommend to use a 1% BSA in PBS solution for blocking as well as for the antibody incubation.

2.) Spectrin is the major constituent of the cytoskeletal network underlying the erythrocyte plasma membrane. Can you therefore please confirm that the source of haemoglobin used is not contaminated with spectrin?

I hope this information is helpful, and I thank you for your cooperation.

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Answer

Thank you for contacting us.

I am sorry to hear you are experiencing difficulties with one of our products. We take product complaints very seriously, and investigate every product that we feel may not be performing correctly.

I am attaching our questionnaire so that we can gather further information regarding the samples tested and the protocol used. Once we receive the completed questionnaire, we will look at the protocol and see if there are any suggestions we can make that may improve the results. This information will also allow us to investigate this case internally and initiate additional testing where necessary. If the product was purchased in the last six months and is being used according to our Abpromise, we would be happy to replace or refund the antibody.


I look forward to receiving your reply.

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Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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